Gel matrix with redox purple for testing and characterizing microorganisms

ABSTRACT

The present invention is directed to kits, methods, and compositions for the characterization of various microorganisms. In particular, the present invention is suited for the characterization of commonly encountered microorganisms (e.g., E. coli, S. aureus, etc.), as well as commercially and industrially important organisms from various and diverse environments. For example, the kits of the present invention are particularly suited for the growth and characterization of the actinomycetes and fungi.

This is a divisional of application Ser. No. 08/762,656, filed Dec. 9,1996 now U.S. Pat. No. 5,882,882 which is a divisional Ser. No.08/421,377 filed Apr. 12, 1995 now U.S. Pat. No. 5,627,045.

FIELD OF THE INVENTION

The present invention relates to growing and testing microorganisms in amultitest format which utilizes a gel forming matrix for the rapidscreening of clinical and environmental cultures. The present inventionis suited for the characterization of commonly encounteredmicroorganisms (e.g., E. coli, S. aureus, etc.), as well as commerciallyand industrially important organisms from various and diverseenvironments (e.g., the present invention is particularly suited for thegrowth and characterization of the actinomycetes and fungi).

BACKGROUND OF THE INVENTION

Ever since the golden age of microbiology in the era of Koch andPasteur, methods for identification of microorganisms have beeninvestigated. Koch's experimental proof that microorganisms causedisease in the early 1800's, provided the impetus to study methods togrow and characterize harmful, as well as beneficial microorganisms.Koch's early experiments to determine the etiology of infectiousdiseases, led to methods for cultivation of microorganisms on thesurface of solid media (e.g., potato slices, see Koch, "Methods for theStudy of Pathogenic Organisms", in T. D. Brock, Milestones inMicrobiology, American Society for Microbiology, 1961, pp. 101-108;originally published as: "Zur Untersuchug von pathogenen Organismen",Mittheilungen aus dem Kaiserlichen Gesundheitsamte 1:1-48 [1881]). Thesestudies eventually led to the development of agar as a culture mediumcomponent useful for producing solid media for growing isolated coloniesof bacteria. To this day, isolated colonies are required (i.e., "purecultures") to biochemically identify organisms.

The field of diagnostic and clinical microbiology has continued toevolve, and yet, there remains a general need for systems that providerapid and reliable biochemical identifications of microorganisms. Inparticular, it has been very difficult to develop an identificationsystem which is capable of identifying various diverse types oforganisms, ranging from the common isolate Escherichia coli to the lesscommonly encountered actinomycetes and fungi.

Methods and identification systems to characterize microorganisms widelyused in industry for production of food and drink (e.g., beer, wine,cheese, yogurt, etc.), the production of antibiotics (e.g., penicillin,streptomycin, etc.), bioremediation of oil spills, biological control ofinsect pests (e.g., Bacillus thuringiensis), and the production ofrecombinant proteins, are still needed. In addition, very fewidentification methods and systems have been developed for environmentaluse and there remains a need for simple and generally usefulidentification methods of many organisms. In particular, methods foridentification and growth of the actinomycetes are lacking.

I. The Actinomycetes

The actinomycetes (members of the order Actinomycetales) include a largevariety of organisms that are grouped together on the basis ofsimilarities in cell wall chemistry, microscopic morphology, andstaining characteristics. Nonetheless, this is a very diverse group oforganisms. For example, genera within this group range from the strictanaerobes to the strict aerobes. Some of these organisms are importantmedical pathogens, while many are saprophytic organisms which benefitthe environment by degrading dead biological or organic matter. Whilemany of these organisms grow optimally at temperatures common in theenvironment (e.g., 25-27° C.), some organisms are quite capable ofgrowing at the body temperature of most mammals (e.g., approximately35-37° C.). However, two genera of medically important actinomycetes(Thermomonospora and Micropolyspora) are true thermophiles, capable ofgrowing at temperatures ranging to 50° C.

Colonies may be bacterium-like (i.e., ranging from butyrous to waxy andglabrous), or fungus-like (i.e., heaped, leathery, membranous coloniesthat are covered with aerial hyphae). Many actinomycetes exhibitfilamentous growth with mycelial colonies, and some actinomycetes causechronic subcutaneous granulomatous abscesses much like those caused byfungi. Because of these similarities, the actinomycetes werelong-regarded as fungi, rather than bacteria (see e.g., G. S. Kobayashi,"Actinomycetes: The fungus-like bacteria", in B. D. Davis et al.,Microbiology, 4th ed., J. B. Lippincott Co., New York, 1990), pp.665-671).

Despite their similarities with the fungi, the actinomycetes havetypical prokaryotic characteristics in terms of nucleoid and cell wallstructure, antimicrobial sensitivity, the absence of sterols, motilityby means of simple flagella, and long filaments of the diameter ofbacteria (approximately 1 μm, compared to the larger fungal hyphae).Microscopically, the morphology of the aerobic actinomycetes varieswidely between genera and species, although they are generally observedas gram-positive rods or branching filaments. Some genera never progressbeyond a typical bacterium-like coccoid or bacillary form (e.g.,Rhodococcus sp.), while others form filaments with extensive branching(e.g., Nocardia, Streptomyces, Actinomadura, and Nocardiopsis). Most arenon-motile in their vegetative phase of growth. However, some generatend to form branched filaments which eventually fragment into motile,flagellated cells (e.g., Oerskovia sp.) (see e.g., G. Land et al.,."Aerobic pathogenic Actinomycetales", in A. Balows et al., Manual ofClinical Microbiology, 1991, pp. 340-359).

Most of the actinomycetes form spores, with the type of spore formationserving as a phylogenetic and taxonomic tool for separating theorganisms into groups. The actinomycetes are highly diverse, with atleast ten subgroups. They are also closely related to such organisms asthe coryneform group (e.g., Corynebacterium sp.), the propionic acidbacteria (e.g., Propionibacterium sp.), and various obligate anaerobes(e.g., Bifidobacterium, Acetobacterium, Butyrvibrio, andThermoanaerobacter). The following table lists the organisms included inthe suprageneric groups of actinomycetes as set forth in the most recentedition of Bergey's Manual, vol. 4, (Stanley T. Williams, editor of vol.4; John G. Holt, editor in chief, Bergey's Manual® of SystematicBacteriology, Williams & Wilkins, pp. 2334-2338 [1989]).

                  TABLE 1                                                         ______________________________________                                        Actinomycetes Groups                                                          Number                                                                              Group         Representative Groups/Genera                              ______________________________________                                        I     Actinobacteria                                                                              Group A: Agromyces,                                                                    Aureobacterium                                                       Group B: Arthrobacter, Rothia                                                 Group C: Cellulomonas, Oerskovia                                              Group D  Actinomyces,                                                                  Arcanobacterium                                                      Group E: Arachnia, Pimelobacter                                               Group F: Brevibacterium                                                       Group G: Dermatophilus                                    II    Actinoplanetes                                                                              Actinoplanes, Ampullanella,                                                   Micromonospora                                            III   Maduromycetes Actindmadura pusilla group,                                                   Microbispora, Streptosporangium                           IV    Micropolysporas                                                                             Actinopolyspora, Faenia,                                                      Saccharomonospora                                         V     Multilocular Sporangia                                                                      Frankia, Geodermatophilus                                 VI    Nocardioforms Nocardia, Rhodococcus, Caseobacter                        VII   Nocardioides  Nocardiodes                                               VIII  Streptomycetes                                                                              Streptomyces, Streptoyerticillium,                                            Kineosporia                                               IX    Thermomonosporas                                                                            Thermomonospora, Nocardiopsis,                                                Actinomadura madurae group                                X     Other Genera  Glycomyces, Kitasatosporia                                                    Spirillospora, Thermoactinomyces                          ______________________________________                                    

Although these organisms may often be identified to the genus levelbased on their morphology at the time of primary isolation, organismsthat have been repeatedly transferred in the laboratory often do notretain their typical morphologic characteristics and must be identifiedbiochemically, or by analysis of their membrane fatty acid composition.Serological methods for identification and differentiation are rarelyused, due to the extensive degree of cross-reactivity among theactinomycetes (see e.g., G. S. Kobayashi, supra, at p. 666).

II. Importance of the Actinomycetes as Pathogens

Many of these organisms are soil-dwellers, with relatively littlepathogenic capabilities. Indeed, the actinomycetes are among the mostabundant of organisms in the soil, where they serve the importantfunction of breaking down proteins, cellulose, and other organic matter.Nonetheless, some Actinomyces, Nocardia, and Streptomyces species areassociated with diseases of medical and veterinary importance,especially in immunocompromised individuals. The spectrum of diseasecaused by the actinomycetes is extremely broad, with pathology that isdependent upon a combination of organism type, tissue involved, and theimmune status of the host. In immunocompetent humans, the most commondiseases are a non-invasive, acute or chronic allergic respiratorysyndrome (e.g., farmer's lung), and mycetoma. In immunocompromisedindividuals, infection often begins in the lung as an acute to chronicsuppurative process, which may progress to cavitation and multi-lobularpulmonary disease. In these patients, infection may spread to otherorgan systems. Importantly, these organisms have a predilection for thecentral nervous system.

Several species of Actinomyces have been associated with actinomycosisin humans and other animals, with A. israelii being the most commonhuman isolate, and A. bovis the most common cattle isolate.Actinomycosis is usually characterized by chronic, destructive abscessesof connective tissues. Abscesses expand into the neighboring tissues andeventually produce burrowing, tortuous sinus tracts to the surface ofthe skin, where purulent material is discharged. In cattle, the lesionsare characteristically large abscesses of the lower jaw (hence thecommon name of the disease, "lumpy jaw"), usually with extensive bonedestruction. As with most saprophytic organisms that occasionally causedisease, actinomycosis is not transmissible from person to person, norbetween humans and other animals. Indeed, it is difficult to establishinfection in laboratory animals.

For in vitro growth in the laboratory, these pathogenic organisms tendto be microaerophilic (e.g., require a decreased oxygen tension foroptimum growth), require rich growth media, optimum incubationtemperatures of 37° C., and about 7 days of incubation. Althoughactinomycetes are soil organisms, actinomycosis is usually caused byendogenous organisms that have colonized the individual, rather thanorganisms from the environment. The organism is usually a commensal,which can be cultured from the tonsils of most humans, and is almostalways present in teeth and gum scrapings. The conditions that lead toinvasiveness are not well characterized, but may be multi-factorial, asactinomycotic infections are often mixed, with various organisms (e.g.,Haemophilus actinomycetemcomitans, Eikenella corrodens, Fusobacterium,and Bacteroides) also present.

In contrast to the Actinomyces, diseases due to Nocardia sp. areassociated with infection of the individual with soil organisms, ratherthan endogenous commensals. Nocardia are among the most clinicallyimportant actinomycetes, as they are responsible for the majority ofdisease associated with this group of organisms. Indeed, the term"nocardiosis" is often used synonymously for pulmonary and disseminatedinfection caused by any of the aerobic actinomycetes (see e.g., G. Landet al., "Aerobic Pathogenic Actinomycetales", in A. Balows et al.,Manual of Clinical Microbiology, 5th ed., American Society forMicrobiology, Washington, D.C., 1991, pages 340-359).

There are two common forms of disease associated with Nocardia sp.,namely, pulmonary nocardiosis resulting from inhalation of the organism,and mycetoma, which is characterized by chronic subcutaneous abscessesresulting from contamination of skin wounds. These infections areusually serious, especially as they are frequently seen in associationwith immunosuppression or chronic underlying diseases (e.g., carcinoma,chronic granulomatous disease, Hodgkin's disease, and leukemia). Onceclinically evident, the progression of nocardiosis tends to beprogressive and fatal, with approximately 50% of patients dying, evenwith aggressive therapy (see e.g., G. S. Kobayashi, "Actinomycetes: TheFungus-Like Bacteria, in B. D. Davis et al. (eds.), Microbiology, 4thed., J. B. Lippincott Co., Philadelphia [1990 ], pages 665-671).

The Nocardia are aerobic organisms which grow on relatively simple mediaover a wide temperature range. As with the mycobacteria, growth inliquid media usually results in the production of a dry, waxy pellicleon the surface of the media. The two species most commonly associatedwith human disease, N. brasiliensis and N. asteroides, share many othercharacteristics with the mycobacteria. For example, they are somewhatacid-fast, more easily stained with fuchsin, and their cell wallscontain components characteristic of mycobacteria and corynebacteria(e.g., mycolic acid residues). Unlike the great majority of organisms,the somewhat harsh methods used to isolate mycobacteria (e.g., treatmentof samples with N-acetyl-L-cysteine, and sodium hydroxide) are oftensuccessful for isolation of Nocardia. Extensive serologiccross-reactions in agglutination and complement fixation tests furtherindicate the relatedness of these groups of organisms.

The Streptomyces are also sometimes associated with actinomycoticabscesses. Mycetomas caused by streptomycetes are clinicallyindistinguishable from those caused by other actinomycetes. However,identification of these organisms can be critical, as they are generallynot susceptible to antimicrobial agents. Therefore, treatment oftenentails surgical removal of the affected area or amputation.

Other members of the actinomycetes are capable of causing disease,including allergic respiratory disease ("farmer's lung"), which occursin agricultural workers who inhale dust from moldy plant material. Thissyndrome has been associated with at least three thermophilicactinomycetes (Thermopolyspora polyspora, Micromonospora vulgaris, andMicropolyspora faeni). This disease is very similar to that caused byinhalation of allergens produced by various fungi, particularlyAspergillus sp.

In addition to the pathogenic potential of this group of organisms,there is also great interest in the particular genera which produceantimicrobial compounds.

III. Industrial Importance of the Actinomycetes

Ever since Waksman isolated actinomycin in 1940, and streptomycin in1943, the streptomycetes have attracted a large amount of attention (seee.g., G. S. Kobayashi, et al., at p. 671). Thousands of soil samplescollected world-wide have resulted in the identification of over 90% ofthe therapeutically useful antibiotics (see e.g., G. S. Kobayashi,"Actinomycetes: The Fungus-Like Bacteria, in B. D. Davis et al. (eds.),Microbiology, 4th ed., J. B. Lippincott Co., Philadelphia [1990], pages665-671). The interest in improving antibiotic qualities and yields hasresulted in various studies on this group of organisms, includingimproved methods for their growth and characterization.

It is important that strains be differentiated in screening programs toidentify antibiotic activities, so that redundant testing is avoided. Inaddition, differentiation facilitates determination of taxonomicrelationships which may lead to other organisms with promisingactivities. Unfortunately, testing of these organisms is often verydifficult. Because they grow as filaments, they have a strong tendencyto form clumps of mycelia which makes them much more difficult tohandle, both in liquid cultures and on solid or semi-solid agar media.Furthermore, because of their complex life cycle which involvessporulation and germination, it is very difficult to obtain cultureswhich perform consistently in metabolic and biochemical testingprograms. In addition, the presence of spores and the potential fortheir inhalation, represents a safety hazard to personnel responsiblefor the cultivation and characterization of these organisms, especiallyin settings where large-scale growth is necessary (e.g., antimicrobialproduction).

These growth characteristics also contribute to the difficultiesassociated with determining the susceptibility of the actinomycetes toantimicrobial compounds. The most frequently used testing methods are amodified Kirby-Bauer disk diffusion method agar dilution, and a minimalinhibitory concentration (MIC) method (see e.g., G. Land et al.,"Aerobic Pathogenic Actinomycetales", in A. Balows et al., Manual ofClinical Microbiology, 5th ed., American Society for Microbiology,Washington, D.C., 1991, at p. 356). However, the success of thesemethods is contingent upon the production of a homogenized suspensionfor use as a standardized inoculum. Most commonly, agitation withsterile glass beads or a tissue homogenizer is used to prepare ahomogenous suspension that can then be diluted to a 0.5 McFarlandstandard prior to inoculating the test media (see e.g., G. Land et al.,"Aerobic Pathogenic Actinomycetales," in A. Balows et al., Manual ofClinical Microbiology, 5th ed., American Society for Microbiology,Washington, D.C., 1991, pages 340-359). These methods involving physicalhomogenization are very labor-intensive and tedious, and they result indamage, fragmentation, and death of some fraction of the cells.Furthermore, the additional manipulation required to produce ahomogenous suspension prior to inoculation increases the risk ofcontamination of laboratory personnel and the laboratory environment.

Therefore, what is needed is a safe, reliable, easy-to-use system forthe characterization and testing of these medically and industriallyimportant organisms. In particular what is need is a rapid method thatis readily automatable and useful in various settings (e.g., clinical,veterinary and environmental laboratories, and industry).

SUMMARY OF THE INVENTION

The present invention relates to growing and testing microorganisms in amultitest format which utilizes a gel forming matrix for the rapidscreening of clinical and environmental cultures. In particular, thepresent invention is suited for the characterization of commonlyencountered microorganisms (e.g., E. coli, S. aureus, etc.), as well ascommercially and industrially important organisms from various anddiverse environments. For example, the present invention is particularlysuited for the growth and characterization of bacteria, as well as theactinomycetes and fungi (e.g., yeasts and molds).

In one embodiment, the present invention provides methods for testingmicroorganisms comprising the steps of: providing a testing meanscomprising redox purple and one or more test substrates; introducingmicroorganisms into the testing means; and detecting the response of themicroorganism to the one or more test substrates. In a preferredembodiment, the testing substrates are selected from the groupconsisting of carbon sources and antimicrobials.

In an alternate embodiment, the testing means further comprises one ormore gel-initiating agents. In a preferred embodiment, thegel-initiating agent comprises cationic salts. In another alternativeembodiment, the testing means further comprises one or more gellingagents. In a preferred embodiment, the microorganisms are in an aqueoussuspension. In another preferred embodiment, the aqueous suspensionfurther comprises one or more gelling agents. It is contemplated thatvarious gelling agents will be used with the present invention,including, but not limited to agar, Gelrite™, carrageenan, and alginicacid.

In one embodiment of the method, the microorganisms are bacteria, whilein another embodiment, the microorganisms are fungi. It is alsocontemplated that the methods of the present invention will be used withmembers of the Order Actinomycetales.

It is contemplated that various testing means will be used in thepresent invention. In one preferred embodiment, the testing meanscomprises at least one microplate, in an alternative embodiment, thetesting means comprises at least one microcard. In yet anotherembodiment, the testing means comprises at least one petri plate.

The present invention also provides a kit, comprising redox purple andone or more test substrates. In a preferred embodiment, the testsubstrates are selected from the group consisting of carbon sources andantimicrobials. In another preferred embodiment, the kit furthercomprises one or more gel-initiating agents. In a particularly preferredembodiment, the gel initiating agent comprises cationic salts. In analternative preferred embodiment, the kit further comprises one or moregelling agents. In another preferred embodiment, the gelling agent isselected from the group consisting of agar, Gelrite™, carrageenan, andalginic acid.

In another embodiment, the kit further comprises a suspension ofmicroorganisms. In one preferred embodiment, the kit further comprises atesting means. It is contemplated that various testing means formatswill be used successfully in various embodiments of the kits of thepresent invention, including microplates, microcards, petri plates, andany other suitable support in which the testing reaction can occur.

In yet another embodiment, the present invention provides a kit,comprising redox purple and one or more gelling agents. It iscontemplated that various gelling agents will be used successfully inthe various embodiments of the kits of the present invention, includingbut not limited to agar, Gelrite™, carrageenan, and alginic acid. In onepreferred embodiment, the kit further comprises one or moregel-initiating agents. In a particularly preferred embodiment, thegel-initiating agent comprises cationic salts. In an alternativeembodiment, the kit further comprises a suspension of microorganisms.

In an alternative embodiment, the kit further comprises one or more testsubstrates. It is contemplated that the test substrates included in thekit of the present invention be selected from the group consisting ofcarbon sources and antimicrobials.

In yet another embodiment, the kit further comprises a testing means. Itis contemplated that various testing means formats will be usedsuccessfully in various embodiments of the kits of the presentinvention, including microplates, microcards, petri plates, and anyother suitable support in which the testing reaction can occur.

The present invention describes test media and methods for the growth,isolation, and presumptive identification of microbial organisms. Thepresent invention contemplates compounds and formulations, as well asmethods particularly suited for the detection and presumptiveidentification of various diverse organisms.

In order to characterize or identify organisms present in a sample, thepresent invention combines a gel-forming suspension with microorganismsthat are already in the form of a pure culture. This is in contrast tothe traditional pour plate method which involves heated agar and asample that contains a mixed culture (see e.g., J. G. Black,Microbiology: Principles and Applications, 2d ed., Prentice Hall,Englewood Cliffs, N.J., p. 153 [1993]; and American Public HealthAssociation, Standard Methods for the Examination of Water andWastewater, 16th ed., APHA, Washington, D.C., pp. 864-866 [1985]).

It is also in contrast to the pour plate method of Roth (U.S. Pat. Nos.4,241,186, and 4,282,317), which utilizes a solidifying pectinsubstance. In the present invention, colloidal gel-forming substancesare used at low concentrations, forming soft gels or viscous colloidalsuspensions that do not need to, and in fact work best, when notcompletely solidified into a rigid gel.

In one embodiment, the present invention provides a method forintroducing microorganisms into a testing device, comprising the stepsof providing a testing device comprising a plurality of testing wells orcompartments, wherein each compartment contains one or moregel-initiating agents; preparing a suspension comprising a pure cultureof microorganisms and an aqueous solution containing a gelling agent,under conditions such that the suspension remains ungelled; andintroducing the suspension into the testing device under conditions suchthat the suspension contacts the gel-initiating agents present in thecompartments and results in the production of a gel or colloidal matrix.

In another embodiment, the present invention provides a method fortesting microorganisms comprising the steps of providing a testingdevice comprising a plurality of testing compartments, wherein thecompartments contain a testing substrate and one or more gel-initiatingagents; preparing a suspension comprising a pure culture ofmicroorganisms and an aqueous solution comprising a gelling agent underconditions such that the suspension remains ungelled; introducing thesuspension into the compartments of the testing device under conditionssuch that the suspension forms a gel matrix within the compartment; anddetecting the response of the microorganisms to the testing substrate.In one preferred embodiment, the testing device is a microplate.

It is contemplated that the microorganisms tested in this method will bebacteria, including members of the Order Actinomycetales, or fungi(e.g., yeasts and molds).

In one embodiment, the gelling agent is selected from the groupconsisting of Gelrite™, carrageenan, and alginic acid. In a particularlypreferred embodiment, the gelling agent is carrageenan which containspredominantly iota-carrageenan. In one embodiment, the gel-initiatingagent comprises cationic salts.

In one embodiment, the testing substrates are selected from the groupconsisting of carbon sources and antimicrobials. In yet anotherembodiment, the method further includes a colorimetric indicator,wherein the colorimetric indicator is selected from the group consistingof chromogenic substrates, oxidation-reduction indicators, and pHindicators.

In yet another embodiment, the present invention encompasses a kit forgrowth and identification of microorganisms comprising: a testing devicecomprising a plurality of testing compartments containing one or moregel-initiating agents; and an aqueous solution comprising a gellingagent. In one preferred embodiment, the testing compartments furthercontain testing substrates, such as carbon sources and antimicrobials.In one embodiment, the gel-initiating agent comprises cationic salts.

In one embodiment of this kit, the testing device is a microplate. In apreferred embodiment, the kit contains a gelling agent that is selectedfrom the group consisting of Gelrite™, carrageenan, and alginic acid. Inone preferred embodiment, the gelling agent is a carrageenan whichpredominantly contains the iota form of carrageenan. In one embodiment,the gel-initiating agent comprises cationic salts.

It is contemplated that the kit of the present invention will be usedwith microorganisms such as bacteria, including members of the OrderActinomycetales, as well as fungi (e.g., yeasts and molds).

It is also contemplated that the kit will also include a colorimetricindicator selected from the group consisting of chromogenic substrates,oxidation-reduction indicators, and pH indicators.

In an alternative embodiment, the present invention comprises a kit forcharacterizing and identifying microorganisms comprising: a microplatetesting device containing a plurality of compartments, wherein thecompartments contain one or more gel-initiating agents and one or moretesting substrates, wherein the testing substrates are selected from thegroup consisting of antimicrobials and carbon sources and an aqueoussuspension comprising a gelling agent.

In one embodiment of this kit, the testing device is a microplate. In apreferred embodiment, the kit contains a gelling agent that is selectedfrom the group consisting of Gelrite™, carrageenan, and alginic acid. Inone preferred embodiment, the gelling agent is a carrageenan whichpredominantly contains the iota form of carrageenan. In one embodiment,the gel-initiating agent comprises cationic salts.

It is contemplated that the kit of the present invention will be usedwith microorganisms such as bacteria, including members of the OrderActinomycetales, as well as fungi (e.g., yeasts and molds).

It is also contemplated that the kit will include a colorimetricindicator selected from the group consisting of chromogenic substrates,oxidation-reduction indicators, and pH indicators.

DESCRIPTION OF THE FIGURES

FIG. 1 is an exploded perspective view of one embodiment of the deviceof the present invention.

FIG. 2 is a top plan view of the device shown in FIG. 1.

FIG. 3 is a cross-sectional view of the device shown in FIG. 2 along thelines of 3--3.

FIG. 4 is a bottom plan view of the device shown in FIG. 1.

FIG. 5 shows the synthesis pathway of redox purple.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the discovery thatvarious organisms contained within a gel matrix may be differentiatedbased on differential biochemical reactions. The present inventionincorporates a multiple test format in a testing device, for presumptiveand rapid microbiological screening of various clinical, veterinary,industrial and environmental specimens. It is also intended that thepresent invention will be useful for definitive identification anddiagnosis. In particular, this invention is suitable for the rapidbiochemical testing of actinomycetes. It is not intended that theinvention be limited to a particular genus, species nor group oforganisms. This medium and method are particularly targeted toward someof the most economically important organisms, as well as species ofclinical importance.

The present invention contemplates an indicator plate similar instructure to microtiter plates ("microplates" or "MicroPlates™")commonly used in the art and commercially available from numerousscientific supply sources (e.g., Biolog, Fisher, etc.). It iscontemplated that the present invention be used with various gellingagents, including but not limited to alginate, carrageenan, and gellangum (e.g., Gelrite™).

Because the organisms are trapped within the gel matrix, the presentinvention is a great improvement over standard microplate testingmethods in which liquid cultures are used. Unlike the liquid format, thegel matrix of the present invention does not spill from the microplate,even if the plate is completely inverted. This safety considerationhighlights the suitability of the present invention for use withorganisms that are easily aerosolized. It is also contemplated that thepresent invention is highly useful in the educational setting, wheresafety is a primary concern. The present invention permits novices towork with bacteria and study their biochemical characteristics with areduced chance of contamination, as compared to other testing systems.

As various organisms may be characterized using the present invention,it is not intended that the choice of primary isolation media be limitedto particular formulae. However, in a preferred embodiment, organismssuch as the actinomyces are grown on an agar medium which stimulates theproduction of aerial conidia. This greatly facilitates the harvesting oforganisms for inoculation in the present invention.

In one embodiment, a microtiter (e.g., microplate) format is used. Inthis embodiment, the gel-forming matrix containing suspendedmicroorganisms is used to inoculate the wells of a microtiter plate oranother receptacle. At the time of inoculation, the gel-forming matrixis in liquid form, allowing for easy dispensing of the suspension intothe compartments. These compartments contain dried biochemicals andcations. Upon contact of the gel-forming matrix with the cations, thesuspension solidifies to form a soft gel, with the organisms evenlydistributed throughout. This gel is sufficiently viscous or rigid thatit will not fall out of the microtiter plate should the plate beinverted.

In another embodiment, a microcard format is used. As shown in FIGS.1-4, one embodiment of the device of the present invention comprises ahousing (100) with a liquid entry port through which the sample isintroduced. The housing further contains a channel (110) providingcommunication to a testing region (120) so that a liquid (not shown) canflow into a plurality of wells or compartments (130). The channel (110)is enclosed by the surface of a hydrophobic, gas-venting membrane (140)adapted for forming one surface of the wells (130) and attached to oneside of the housing (100). The housing (100) can be sealed on its otherside by a solid base (150). In other embodiments, a flexible tape (notshown) may be substituted for the solid base (150) or the solid base(150) may be molded so as to be integral with the housing (100).

After filling the device with the gel-forming matrix containingmicroorganisms, (not shown) an optional non-venting material such astape (160) can be adhered to the outer surface of the gas-ventingmembrane (140) to seal it against evaporation of the gel matrix withinthe device through the gas-venting membrane. At the time of delivery,the gel-forming matrix with suspended organisms is in liquid form. Oncethe liquid comes into contact with the compounds present in the testingregion, a gel matrix is produced, trapping the suspended microorganisms.

Although embodiments have been described with some particularity, manymodifications and variations of the preferred embodiment are possiblewithout deviating from the invention.

Definitions

The terms "sample" and "specimen" in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, and saliva, as well as solidtissue. These terms also refers to swabs and other sampling deviceswhich are commonly used to obtain samples for culture of microorganisms.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Environmental samples include environmentalmaterial such as surface matter, soil, water, and industrial samples, aswell as samples obtained from food and dairy processing instruments,apparatus, equipment, disposable, and non-disposable items. Theseexamples are not to be construed as limiting the sample types applicableto the present invention.

Whether biological or environmental, a sample suspected of containingmicroorganisms may (or may not) first be subjected to an enrichmentmeans to create a "pure culture" of microorganisms. By "enrichmentmeans" or "enrichment treatment", the present invention contemplates (i)conventional techniques for isolating a particular microorganism ofinterest away from other microorganisms by means of liquid, solid,semi-solid or any other culture medium and/or technique, and (ii) noveltechniques for isolating particular microorganisms away from othermicroorganisms. It is not intended that the present invention be limitedonly to one enrichment step or type of enrichment means. For example, itis within the scope of the present invention, following subjecting asample to a conventional enrichment means, to subject the resultantpreparation to further purification such that a pure culture of a strainof a species of interest is produced. This pure culture may then beanalyzed by the medium and method of the present invention.

As used herein, the term "culture" refers to any sample or specimenwhich is suspected of containing one or more microorganisms. "Purecultures" are cultures in which the organisms present are only of onestrain of a particular genus and species. This is in contrast to "mixedcultures", which are cultures in which more than one genus and/orspecies of microorganism are present.

As used herein, the term "organism" is used to refer to any species ortype of microorganism, including but not limited to bacteria, yeasts andother fungi. As used herein, the term fungi, is used in reference toeukaryotic organisms such as the molds and yeasts, including dimorphicfungi.

As used herein, the term "spore" refers to any form of reproductiveelements produced asexually (e.g., conidia) or sexually by suchorganisms as bacteria, fungi, algae, protozoa, etc. It is also used inreference to structures within microorganisms such as members of thegenus Bacillus, which provide advantages to the individual cells interms of survival under harsh environmental conditions. It is notintended that the term be limited to any particular type or location ofspores, such as "endospores" or "exospores". Rather, the term is used inthe very broadest sense.

As used herein, the terms "microbiological media" and "culture media",and "media" refer to any substrate for the growth and reproduction ofmicroorganisms. "Media" may be used in reference to solid plated mediawhich support the growth of microorganisms. Also included within thisdefinition are semi-solid and liquid microbial growth systems includingthose that incorporate living host organisms, as well as any type ofmedia.

As used herein, the term "carbon source" is used in reference to anycompound which may be utilized as a source of carbon for bacterialgrowth and/or metabolism. Carbon sources may be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, and peptides.

As used herein, the term "nitrogen source" is used in reference to anycompound which may be utilized as a source of nitrogen for bacterialgrowth and/or metabolism. As with carbon sources, nitrogen sources maybe in various forms, such as free nitrogen, as well as compounds whichcontain nitrogen, including but not limited to amino acids, peptones,vitamins, and nitrogenous salts.

As used herein, the term "antimicrobial" is used in reference to anycompound which inhibits the growth of, or kills microorganisms. It isintended that the term be used in its broadest sense, and includes, butis not limited to compounds such as antibiotics which are producednaturally or synthetically. It is also intended that the term includescompounds and elements that are useful for inhibiting the growth of, orkilling microorganisms.

As used herein, the term "testing substrate" is used in reference to anycarbon and/or nitrogen source that may be utilized to differentiatebacteria based on biochemical characteristics. For example, onebacterial species may utilize one testing substrate that is not utilizedby another species. This utilization may then be used to differentiatebetween these two species. It is contemplated that numerous testingsubstrates be utilized in combination. Testing substrates may be testedindividually (e.g., one substrate per testing well or compartment, ortesting area) or in combination (e.g., multiple testing substrates mixedtogether and provided as a "cocktail").

Following exposure to a testing substrate such as a carbon or nitrogensource, or an antimicrobial, the response of an organism may bedetected. This detection may be visual (i.e., by eye) or accomplishedwith the assistance of machine(s) (e.g., the Biolog MicroStationReader™). For example, the response of organisms to carbon sources maybe detected as turbidity in the suspension due to the utilization of thetesting substrate by the organisms. Likewise, growth can be used as anindicator that an organism is not inhibited by certain antimicrobials.In one embodiment, color is used to indicate the presence or absence oforganism growth/metabolism.

As used herein, the terms "chromogenic compound" and "chromogenicsubstrate", refer to any compound useful in detection systems by theirlight absorption or emission characteristics. The term is intended toencompass any enzymatic cleavage products, soluble, as well asinsoluble, which are detectable either visually or with opticalmachinery. Included within the designation "chromogenic" are allenzymatic substrates which produce an end product which is detectable asa color change. This includes, but is not limited to any color, as usedin the traditional sense of "colors," such as indigo, blue, red, yellow,green, orange, brown, etc., as well as fluorochromic or fluorogeniccompounds, which produce colors detectable with fluorescence (e.g., theyellow-green of fluorescein, the red of rhodamine, etc.). It is intendedthat such other indicators as dyes (e.g., pH) and luminogenic compoundsbe encompassed within this definition.

As used herein, the commonly used meaning of the terms "pH indicator,""redox indicator," and "oxidation-reduction indicator," are intended.Thus, "pH indicator" encompasses all compounds commonly used fordetection of pH changes, including, but not limited to phenol red,neutral red, bromthymol blue, bromcresol purple, bromcresol green,bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple,xylenol blue, methyl red, methyl orange, and cresol red. The terms"redox indicator" and "oxidation-reduction indicator" encompass allcompounds commonly used for detection of oxidation/reduction potentials(i.e., "eH" ) including, but not limited to various types or forms oftetrazolium, resazurin, methylene blue, and quinone-imide redox dyesincluding the compounds known as "methyl purple" and derivatives ofmethyl purple. The quinone-imide redox dye known as methyl purple isreferred to herein as "redox purple." In a particularly preferredembodiment, "redox purple" comprises the compound with the chemicalstructure shown in FIG. 5, VI. It is contemplated that analogousderivatives of the reagent (e.g., alkali salts, alkyl O-esters), withmodified properties (e.g., solubility, cell permeability, toxicity,and/or modified color(s)/absorption wavelengths) will be produced usingslight modifications of the methods described in Example 13. It is alsocontemplated that various forms of redox purple (e.g., salts, etc.), maybe effectively used in combination as a redox indicator in the presentinvention.

As used herein, the terms "testing means" and "testing device" are usedin reference to testing systems in which at least one organism is testedfor at least one characteristic, such as utilization of a particularcarbon source, nitrogen source, or chromogenic substrate, and/orsusceptibility to an antimicrobial agent. This definition is intended toencompass any suitable means to contain a reaction mixture, suspension,or test. It is intended that the term encompass microtiter plates, petriplates, microcard devices, or any other supporting structure that issuitable for use. For example, a microtiter plate having at least onegel-initiating agent included in each of a plurality of wells orcompartments, comprises a testing means. Other examples of testing meansinclude microtiter plates without gel-initiating means included in thewell. It is also intended that other compounds such as carbon sources orantimicrobials will be included within the compartments. The definitionis also intended to encompass a "microcard" or miniaturized plates orcards which are similar in function, but much smaller than standardmicrotiter plates (for example, many testing devices can be convenientlyheld in a user's hand). It is not intended that the present invention belimited to a particular size or configuration of testing device ortesting means. For example, it is contemplated that various formats willbe used with the present invention, including, but not limited tomicrotiter plates, microcards, petri plates, petri plates with internaldividers used to separate different media placed within the plate, testtubes, as well as many other formats.

As used herein, the term "gelling agent" is used in a broad genericsense, and includes compounds that are obtained from natural sources, aswell as those that are prepared synthetically. As used herein, the termrefers to any substance which becomes at least partially solidified whencertain conditions are met. For example, one gelling agent encompassedwithin this definition is Gelrite™, a gellan which forms a gel uponexposure to divalent cations (e.g., Mg² + or Ca²⁺). Gelrite™ is producedby deacetylating a natural polysaccharide produced by Pseudomonaselodea, and is described by Kang et al. (U.S. Pat. Nos. 4,326,052 and4,326,053, herein incorporated by reference).

Included within the definition are various gelling agents obtained fromnatural sources, including protein-based as well as carbohydrate-basedgelling agents. One example is bacteriological agar, a polysaccharidecomplex extracted from kelp. Also included within the definition aresuch compounds as gelatins (e.g., water-soluble mixtures of highmolecular weight proteins obtained from collagen), pectin (e.g.,polysaccharides obtained from plants), carrageenans and alginic acids(e.g., polysaccharides obtained from seaweed), and gum (e.g.,mucilaginous excretions from some plants and bacteria). It iscontemplated that various carrageenan preparations will be used in thepresent invention, with iota carrageenan comprising a preferredembodiment. It is also contemplated that gelling agents used in thepresent invention may be obtained commercially from a supply company,such as Difco, BBL, Oxoid, Marcor, Sigma, or any other source.

It is not intended that the term "gelling agent" be limited to compoundswhich result in the formation of a hard gel substance. A spectrum iscontemplated, ranging from merely a more thickened or viscous colloidalsuspension to one that is a firm gel. It is also not intended that thepresent invention be limited to the time it takes for the suspension togel.

Importantly, it is intended that the present invention provides agelling agent suitable for production of a matrix in which organisms maygrow (i.e., a "gel matrix"). The gel matrix of the present invention isa colloidal-type suspension of organisms produced when organisms aremixed with an aqueous solution containing a gelling agent, and thissuspension is exposed to a gel-initiating agent. It is intended thatthis colloidal-type gel suspension be a continuous matrix mediumthroughout which organisms may be evenly dispersed without settling outof the matrix due to the influence of gravity. The gel matrix mustsupport the growth of organisms within, under, and on top of the gelsuspension.

As used herein the term "gel-initiating agent" refers to any compound orelement which results in the formation of a gel matrix, followingexposure of a gelling agent to certain conditions or reagents. It isintended that "gel-initiating agent" encompass such reagents as cations(e.g., Ca²⁺, Mg²⁺, and K⁺). Until the gelling agent contacts at leastone gel-initiating agent, any suspension containing the gelling agentremains "ungelled" (i.e., there is no thickening, increased viscosity,nor hardening of the suspension). After contact, the suspension willbecome more viscous and may or may not form a rigid gel (i.e., contactwill produce "gelling").

As used herein, the term "inoculating suspension" or "inoculant" is usedin reference to a suspension which may be inoculated with organisms tobe tested. It is not intended that the term "inoculating suspension" belimited to a particular fluid or liquid substance. For example,inoculating suspensions may be comprised of water, saline, or an aqueoussolution which includes at least one gelling agent. It is alsocontemplated that an inoculating suspension may include a component towhich water, saline or any aqueous material is added. It is contemplatedin one embodiment, that the component comprises at least one componentuseful for the intended microorganism. It is not intended that thepresent invention be limited to a particular component.

As used herein, the term "kit" is used in reference to a combination ofreagents and other materials. It is contemplated that the kit mayinclude reagents such as carbon sources, nitrogen sources, chromogenicsubstrates, antimicrobials, diluents and other aqueous solutions, aswell as microplates (e.g., GN, GP, YT, SF-N, SF-P, and otherMicroPlates™, obtained from Biolog), inoculants, microcards, and platedagar media. The present invention contemplates other reagents useful forthe growth, identification and/or determination of the antimicrobialsusceptibility of microorganisms. For example, the kit may includereagents for detecting the growth of microorganisms followinginoculation of kit components (e.g.,tetrazolium or resazurin included insome embodiments of the present invention). It is not intended that theterm "kit" be limited to a particular combination of reagents and/orother materials. Further, in contrast to methods and kits which involveinoculating organisms on or into a preformed matrix such as an agarsurface or broth, the present invention involves inoculation of atesting plate in which the organisms are suspended within a gel-formingmatrix.

As used herein, the term "primary isolation" refers to the process ofculturing organisms directly from a sample. Thus, primary isolationinvolves such processes as inoculating an agar plate from a cultureswab, urine sample, environmental sample, etc. Primary isolation may beaccomplished using solid or semi-solid agar media, or in liquid. As usedherein, the term "isolation" refers to any cultivation of organisms,whether it be primary isolation or any subsequent cultivation, including"passage" or "transfer" of stock cultures of organisms for maintenanceand/or use.

As used herein, the term "presumptive diagnosis" refers to a preliminarydiagnosis which gives some guidance to the treating physician as to theetiologic organism involved in the patient's disease. Presumptivediagnoses are often based on "presumptive identifications," which asused herein refer to the preliminary identification of a microorganismbased on observation such as colony characteristics, growth on primaryisolation media, gram stain results, etc.

As used herein, the term "definitive diagnosis" is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. The term "definitive identification" is used inreference to the final identification of an organism to the genus and/orspecies level.

GENERAL DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the discovery thatvarious organisms may be identified and differentiated based ondifferential biochemical reactions observed in gelled media. Themultiple test medium of the present invention permits presumptive andrapid microbiological screening of various specimens. In particular,this invention in the form of a kit, is suitable for the easy and rapidbiochemical testing of various microorganisms, including theactinomycetes and fungi.

The present invention also contemplates a multitest indicator plate thatis generally useful in the identification and antimicrobial sensitivitytesting of microorganisms. This medium and method are particularlytargeted toward some of the most economically important organisms, aswell as species of clinical importance. It is not intended that theinvention be limited to a particular genus, species nor group oforganisms.

It is contemplated that the present invention be used with variousgelling agents, including, but not limited to agar, pectin, alginate,alginic acid, silica, gellans and gum. In one embodiment, the pectinmedium of Roth (U.S. Pat. Nos. 4,241,186, and 4,282,317; hereinincorporated by reference) is used. However, this is not a preferredembodiment, as pectin is not a colorless compound itself. In onepreferred embodiment, the gellan of Kang et al. (U.S. Pat. Nos.4,326,052 and 4,326,053, herein incorporated by reference) is used. Inanother preferred embodiment, carrageenan is used as the gelling agent.In a particularly preferred embodiment, carrageenan type II or anycarrageenan which contains predominantly the iota form of carrageenan isused. In each embodiment, the microorganisms to be tested are mixed in asuspension comprising a gelling agent, and then inoculated into a well,compartment, or other receptacle, which contains the biochemical(s) tobe tested, along with a gel-initiating agent such as various cations.Upon contact of the gelling agent with the gel-initiating agent (e.g.,cations), the suspension solidifies to form a viscous colloid or gel,with the organisms evenly distributed throughout.

The present invention contemplates a testing device that is a microplatesimilar in structure to microtiter plates ("microplates" or"MicroPlates™") commonly used in the art and commercially available fromnumerous scientific supply sources (e.g., Biolog, Fisher, etc.). Thus,in one embodiment, standard 96-well microtiter plates are used.

An alternate embodiment of the invention generally relates to a"microcard" device for the multiparameter testing of chemical,biochemical, immunological, biomedical, or microbiological samples inliquid or liquid suspension form in a small, closed, easy-to-filldevice, and is particular suitable for multiparameter testing andidentification of microorganisms. It is not intended that the presentinvention be limited to a particular sized device. Rather, thisdefinition is intended to encompass any device smaller than the commonlyused, 96-well microplates. In one particularly preferred embodiment, themicrocard is approximately 75 mm in width and 75 mm in length, andapproximately 3 mm in depth. Approximately one-tenth the volume of cellsare used to inoculate the compartments of the device, as compared tostandard microplates. Indeed, the present invention contemplates adevice comprising: a) a housing; b) a testing region contained withinthe housing; c) a liquid receiving means on an external surface of thehousing; d) a liquid flow-directing means providing liquid communicationbetween the testing region and the liquid receiving means; and e) agas-venting, liquid barrier in fluidic communication with the testingregion.

After the device has been filled, a non-venting, sealing tape can beapplied to the device to cover the gas-venting, liquid barrier to reducethe evaporation of the liquid from the device; the tape can permit themolecular diffusion of oxygen into or out of the device to maintain thedesired chemical or biochemical environment within the device forsuccessful performance of the test. Where the liquid receiving meanscomprises liquid entry ports, a similar closing tape can be applied toclose the port or ports to prevent spilling and evaporation of theliquid therefrom.

With any of the testing formats, the visual result that is detected byeye or by instrument can be any optically perceptible change such as achange in turbidity, a change in color, or the emission of light, suchas by chemiluminescence, bioluminescence, or by Stokes shift. Colorindicators may be, but are not limited to, redox indicators (e.g.,tetrazolium, resazurin, and/or redox purple), pH indicators, or variousdyes and the like. Various dyes are described in U.S. Pat. Nos.4,129,483, 4,235,964 and 5,134,063 to Barry R. Bochner, herebyincorporated by reference. See also B. R. Bochner, Nature 339:157(1989); and B. R. Bochner, ASM News 55:536 (1990). A generalizedindicator useful for practice of the present invention is also describedby Bochner and Savageau. See B. Bochner and M. Savageau, Appl. Environ.Microbiol., 33:434 (1977).

Testing based on the redox technology is extremely easy and convenientto perform. A cell suspension is prepared and introduced into thetesting compartments of the device. Each compartment is prefilled with adifferent substrate.

In a preferred embodiment, all wells are prefilled with test formulacomprising a basal medium that provides nutrients for themicroorganisms, and a color-change indicator, and each compartment isprefilled with a different carbon compound or "testing substrate,"against which the microorganism is tested. "Basal medium," as usedherein, refers to a medium which provides nutrients for themicroorganisms, but does not contain sufficient concentrations of carboncompounds to trigger a color response from the indicator. "Carboncompound," "carbon source" and "testing substrate" are equivalent terms,and are used interchangeably herein to refer to a carbon chemical insufficient concentration as to trigger a color response from theindicator when it is utilized (metabolized) by a microorganism (e.g.,GN, GP, YT, and other MicroPlates™ commercially available from Biolog).In a particularly preferred embodiment, redox purple is used as a redoxindicator in the present invention.

One of the principal uses of the present invention is as a method anddevice for simple testing and speciation of microorganisms. The presentinvention contemplates microbiological testing based on the redoxtechnology discussed above wherein a sample of a pure culture ofmicroorganism is removed from a culture medium on which it has beengrown and suspended in saline or water at a desired density. Thissuspension is then introduced into the compartments of the testingdevice which have been prefilled with basal medium, indicator, andsubstrate chemicals. The method is extremely easy and convenient toperform, and, unlike other approaches, the method and device do notrequire skilled personnel and cumbersome equipment.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); TSA (trypticase soy agar); YME or YEME (Yeastextract-malt extract agar); EMB (eosin methylene blue medium); MacConkey(MacConkey medium); Redigel (RCR Scientific, Goshen, Ind.); Gelrite™(Merck and Co., Rahway, N.J., available from Sigma); Remel, (Remel,Lenexa, Kans.); Oxoid (Oxoid, Basingstoke, England); BBL (BectonDickinson Microbiology Systems, Cockeysville, Md.); DIFCO (DifcoLaboratories, Detroit, Mich.); U.S. Biochemical (U.S. Biochemical Corp.,Cleveland, Ohio); Fisher (Fisher Scientific, Pittsburgh, Pa.); Sigma(Sigma Chemical Co., St. Louis, Mo.); Biolog (Biolog, Inc., Hayward,Calif.); ATCC (American Type Culture Collection, Rockville, Md.); CBS(Centraalbureau Voor Schimmelcultures, Delft, Netherlands); CCUG(Culture Collection of University of Gothenberg, Gothenberg, Sweden);GSU (Georgia State University, Atlanta, Ga.); NRRL (USDA NorthernRegional Research Laboratory, Peoria, Ill.); and NCYC (NationalCollection of Yeast Cultures, Norwich, England); NCCLS (NationalCommittee for Clinical Laboratory Standards); API (API AnalytabProducts, Plainview, N.Y.); Flow (Flow Laboratories, McLean, Va.);Biomerieux (Biomerieux, Hazelwood, Mo.).

The following Tables list the principal bacterial strains used in thefollowing Examples, with Table 2 listing the various actinomycetes, andTable 3 listing other species of microorganisms.

                  TABLE 2                                                         ______________________________________                                        Actinomycetes Tested                                                          Organism             Source and Number                                        ______________________________________                                        Actinomadura ferruginea                                                                            USDA                                                                          NRRL B-16096                                             Actinoplanes rectilineatus                                                                         USDA                                                                          NRRL B-16090                                             Micromonospora chalcea                                                                             USDA                                                                          NRRL B-2344                                              Norcardiopsis dassonvillei                                                                         USDA                                                                          NRRL B-5397                                              Saccharopolyspora hirsuta                                                                          USDA                                                                          NRRL B-5792                                              Streptomyces albidoflavus                                                                          USDA                                                                          NRRL B-1271                                              Streptomyces coeruleoribidus                                                                       USDA                                                                          NRRL B-2569                                              Streptomyces griseus USDA                                                                          NRRL B-2682                                              Streptomyces hygroscopicus                                                                         USDA                                                                          NRRL B-1477                                              Streptomyces lavendulae                                                                            USDA                                                                          NRRL B-1230                                              Streptoverticillium solmonis                                                                       USDA                                                                          NRRL B-1484                                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Other Organisms Tested                                                        Organism             Source and Number                                        ______________________________________                                        Escherichia coli     ATCC #25922                                              Staphylococcus aureus                                                                              ATCC #29213                                              Providencia stuartii ATCC #33672                                              Pseudomonas cepacta  ATCC #25416                                              Neisseria lactamica  CCUG#796                                                 Xanthomonas maltophilia                                                                            ATCC#13637                                               Vibrio metschnikovii ATCC#7708                                                Cedecea neteri       ATCC#18763                                               Rhodococcus equi     ATCC#6939                                                Dipodascus ovetensis ATCC #10678                                              Cryptococcus laurentii                                                                             CBS#139                                                  Cryptococcus terreus A                                                                             CBS#1895                                                 Kluyveromyces marxianus                                                                            GSU#C90006070                                            Saccharomyces cerevisiae A                                                                         NCYC##505                                                Williopsis saturnus var. saturnus                                                                  GSU#WC-37                                                Penicillium notatum  ATCC#9179                                                Penicillium chrysogenum                                                                            ATCC#11710                                               Rhizomucor pusillus  ATCC#32627                                               Aspergillus niger    ATCC#16404                                               Tricophyton mentagrophytes                                                                         ATCC#9129                                                ______________________________________                                    

EXAMPLE 1 Primary Growth of Actinomycetes

In this example, several attempts were made to grow variousactinomycetes in R2A liquid media prepared from the recipe of Reasonerand Geldreich (Reasoner and Geldreich, Appl. Environ. Microbiol., 49:1-7[1985]), prior to preparation of inoculum suspensions for inoculatingcommercially available MicroPlates™ from Biolog (Biolog's GN, GP, and YTMicroPlates™). This method proved unsuccessful and cumbersome. Also, itwas virtually impossible to obtain uniform (homogenous) cultures ofsatisfactory quality.

Next, these organisms were grown on the surface of various agar media.It was thought this might provide a very simple means to harvest sporesfrom the culture, as the colonies tend to anchor into the agar matrixitself. The media used in this example included Sporulation Agar(described by R. Atlas in Handbook of Microbiological Media, CRC Press,Boca Raton, Fla., p. 834 [1993]), and YEME Agar with glucose omitted(described by E. B. Shirling and D. Gottlieb, in "Methods forCharacterization of Streptomyces Species," Int'l J. System. Bacteriol.,16:313-330 [1966])(hereinafter referred to as YEMEWG).

Sporulation Agar (also known as m-Sporulation Agar) comprises agar (15g/l), glucose (10 g/l), tryptose (2 g/l), yeast extract (1 g/l), beefextract (1 g/l), and FeSO₄.7H₂ O (1 μg/l), pH 7.2±0.2 at 25° C. Theseingredients are added to 1 liter of distilled/deionized water, and mixedthoroughly with heat to boiling. After the mixture has dissolved, it isautoclaved at 15 psi (121° C.) for 15 minutes, and dispensed intoplates.

YEMEWG Agar comprises Bacto yeast extract (4 g/l; Difco), and Bacto-maltextract (10 g/l; Difco). These ingredients are added to 1 liter ofdistilled/deionized water and mixed thoroughly. The pH is adjusted to7.3, and agar (20 g/l) is added to the mixture. The mixture is thenautoclaved at 121° C. for 15-20 minutes, and dispensed into Petri platesafter it is sufficiently cooled. YEMEWG was used because preliminarystudies indicated that, while glucose-containing YEME agar was adequatefor growth of the Streptomyces species, genera such as Nocardiopsis andActinoplanes grew better when glucose was omitted from the mediumrecipe.

Because of the interest in obtaining spores, media that encouragesporulation were tried. For example, YEMEWG was found to be particularlyuseful, as this medium gave satisfactory growth and sporulation of moststrains tested within 2-4 days of incubation at 26° C. Various agarconcentrations were tested during these preliminary studies, and it wasfurther observed that when YEMEWG was used, improved sporulationoccurred in the presence of a higher agar concentration (e.g., 25 g/l,rather than the 15 g/l, traditionally used in microbiological agarmedia).

This approach of growing actinomycetes on a sporulation-inducing mediumwould have the additional benefit of standardizing the physiologicalstate of the organisms, and would permit preparation of inoculaprimarily from spheroidal spores. It was usually a relatively simplematter to produce uniform, homogeneous suspensions containing spores.Occasionally, however, large clumps of the organisms and their aerialmycelia are obtained which do not readily disperse in solution. Whenclumps are formed, the suspension is allowed to sit for a few minutes,permitting the large fragments to settle to the bottom of the tube. Useof a light inoculum (i.e., a 1:10 dilution of an initial suspensionwhere the initial suspension has a transmittance level of 70%) alsohelps avoid problems with clumping of large fragments. Therefore, clumpscan be avoided in the preparation of the final inoculum because only asmall, clump-free aliquot of the initial suspension is used. For thoseorganisms that sporulate poorly, fragments of rods and/or mycelialfilaments were obtained from the agar surface in the same manner.

This example highlights the advantages of the present invention for theprimary growth and subsequent characterization of actinomycetes, incontrast to references that indicate growth of actinomycetes is veryslow. For example, Bergey's Manual® (T. Cross, "Growth and Examinationof Actinomycetes--Some Guidelines," in J. Holt et al., "TheActinomycetes," Bergey's Manual® of Determinative Bacteriology, 9th ed.,Williams & Wilkins, Baltimore, pp. 605-609 [1994]) indicates that"mature aerial mycelium with spores may take 7-14 days to develop, andsome very slow-growing strains may require up to 1 month's incubation."This is in stark contrast to the present invention, in which heavygrowth and sporulation is achieved within 2-4 days of incubation.

EXAMPLE 2

Preparation of Inoculum

In this experiment, a method more optimal for preparation of ahomogeneous inoculum was determined. For example, it was found that aneasy and reproducible method was to grow the organisms as described inExample 1 on YEMEWG-prepared with 25 g/l agar, or other suitable agarmedium. A low density inoculum (i.e., 0.01 to 0.1 OD₅₉₀) was thenprepared by moistening a cotton swab and rubbing it across the top ofthe colonies to harvest mycelia and spores. It was determined thatsterilized water and 0.85% sterile saline worked reasonably well as asuspension medium for all strains. However, some strains exhibited apreference for one or the other. For example, Streptomycescoeruleoribidus, S. hygroscopicus, and S. albidoflavus produced anaverage of ten additional positive reactions when water was used as thesuspension medium, whereas thirteen additional positive reactions wereobserved for S. lavendulae when saline was used as the suspensionmedium. The majority of the Actinomycetes performed better when waterwas used. Therefore, water was used routinely to prepare thesuspensions.

EXAMPLE 3

Preparation of Multi-Test Plates

The inocula prepared as described in Example 2 were used to inoculatevarious Biolog MicroPlates™, including the commercially available GN,GP, and YT MicroPlates™. A few strains worked well upon inoculation intothe GN or GP MicroPlates™ (e.g., S. lavendulae). However, for moststrains (e.g., A. ferruginea, and N. dassonvillei) no positive reactionswere observed. In addition, positive reactions were observed in all ofthe test wells for some organisms (e.g., S. hirsuta), indicating thatthere was a problem with false positive results.

Much improved results were obtained when the wells located in the bottomfive rows of the YT MicroPlate™ were used. It was thought that thisobservation was due to the absence of tetrazolium in these wells, as thetetrazolium present in the other wells appeared to inhibit the growth ofthe organisms. This was confirmed by testing the ability of theorganisms to grow on YEMEWG agar media containing various concentrationsof tetrazolium (20, 40, 60 and 80 mg/l). Many strains (e.g., S.coeruleoribidus, S. hygroscopicus, S. lavendulae, M chalcea, N.dassonvillei, and A. rectilineatus) were inhibited at all of thesetetrazolium concentrations. Other organisms, such as S. griseus, S.albidoflavus, and S. hirsuta, were somewhat inhibited at the highertetrazolium concentrations, but grew in tetrazolium concentrations of 20and 40 mg/l.

Based on these experiments, MicroPlate™ were then tested that containedno tetrazolium (e.g., "SF-N" [GN MicroPlate™ without tetrazolium] and"SF-P" [GP MicroPlate™ without tetrazolium] MicroPlates™). These plateswere inoculated with water or saline suspensions of variousactinomycetes, and incubated at 26° C. for 1-4 days. Increased turbidity(i.e., growth of the organisms) was readable visually, or with amicroplate reader (e.g., a Biolog MicroStation Reader™, commerciallyavailable from Biolog), in as little as 24 hours for some strains. Forthe slow growing strains, growth was readable and the resultsinterpretable within 3-4 days, representing a significant improvementover the 7-10 day incubation period required using routine methods.

EXAMPLE 4 Use Of Gelrite™

Although growth was observable in the multi-test system described inExample 3, the results were still not completely satisfactory, due tothe unique growth characteristics of the actinomycetes. Many of thesestrains adhered to the plastic walls of the microplate wells, therebymaking detection of increased turbidity less than optimal. When theinoculating suspension is a liquid, turbidity often was concentratedalong the outer circumference of the wells, rather than producing auniform dispersion of turbidity throughout the wells.

In order to facilitate uniform dispersion of the inoculating suspensioncontaining organisms throughout the well, a gelling agent was added tothe suspension to prevent individual cells from migrating to the wellwalls. For example, preparations of Gelrite™ (commercially availablefrom Sigma, under this name, as well as "Phytagel") were found to behighly satisfactory. Gelrite™ does not form a gel matrix until it isexposed to gel-initiating agents, in particular, positively charged ionssuch as divalent cations (e.g., Mg²⁺ and Ca²⁺). As soon as the Gelrite™comes into contact with the salts present in the bottom of themicroplate wells, the gelling reaction begins and results in theformation of a gel matrix within a few seconds.

Various concentrations of Gelrite™ were tested, including 0.1, 0.2, 0.3,0.4, 0.5 and 0.6%. All concentrations gelled in the microplate, with thehigher concentrations producing a harder gel.

In view of the fact that most of the actinomycetes are obligate aerobes,there was a concern that the oxygen concentration within the gel must besufficient to permit growth. Thus, various gel depths were tested byusing 50, 100, or 150 μl suspensions of organisms in the wells. Each ofthese depths resulted in good growth of organisms, although it wasobserved that 0.4% Gelrite™ and an inoculum of 100 μl produced optimalresults, even with organisms such as Streptomyces lavendulae, a speciesthat is strongly hydrophobic and clings to the walls of wells when it issuspended in water. The 0.4% concentration of Gelrite™ was found toproduce an appropriate degree of viscosity to readily permit preparationof microbial suspensions and still be easily pipetted.

The entire procedure for growth and testing of the actinomycetesrequired a total of 3-7 days, including primary inoculation on YEMEWGmedium and other suitable media to determination and analysis of thefinal results. Importantly, a minimum amount of personnel time wasrequired (i.e., just the few minutes necessary to inoculate the primarygrowth medium and then prepare the suspension for biochemical testing).Thus, the present invention provides a much improved means for the rapidand reliable identification of actinomycetes.

EXAMPLE 5 Comparison of Water and Gelrite™

In this Example, the eleven actinomycetes listed in Table 2 were testedin both water and gel suspensions. For each organism, a water suspensionof organisms with an optical transmittance of 70%, was diluted 1:10 ineither water or 0.4% Gelrite™. Thus, two samples of each organism wereproduced, one sample being a water suspension and one sample being asuspension which included Gelrite™.

One hundred microliters of each sample were inoculated into SF-PMicroPlates™ (GP MicroPlates™ without tetrazolium; commerciallyavailable from Biolog). The MicroPlates™ were incubated at 27° C. for 48hours, and observed for growth. As shown in the table below, the numberof positive reactions increased dramatically for the organisms suspendedin Gelrite™, as compared to water.

                  TABLE 4                                                         ______________________________________                                        Growth of Selected Streptomyces Species                                                    Number of  Number or                                                          Positive/Borderline                                                                      Positive/Borderline                                                Reactions in                                                                             Reactions in                                                       Water Suspensions                                                                        Gel Suspensions                                                    (+/b)      (+/b)                                                 ______________________________________                                        Streptomyces coeruleorubidus                                                                 5/35         35/25                                             Streptomyces griseus                                                                         30/15        43/12                                             Streptomyces lavendulae                                                                      8/18         24/12                                             ______________________________________                                    

EXAMPLE 6 Use of Resazurin

In this Example, three concentrations of resazurin dye (25 mg/l, 50mg/l, and 75 mg/l) were used as a redox color indicator of organismgrowth and metabolism. All of the eleven actinomycete strains listed inTable 2 were tested using these three concentrations of resazurin, and0.4% Gelrite™.

The expected color reaction, a change from blue to pink and eventuallyto colorless, as the dye is progressively reduced, occurred with alltest organisms after 48 hours of incubation at 27° C. This observationprovides a supplemental indicator of organism metabolism in addition toturbidity. No single resazurin concentration provided uniformly optimalresults. For example, N. dassonvillei produced a good differentialpattern of color change at 25 mg/l and 50 mg/l, whereas S. lavendulaeproduced false positive results (i.e., all colorless wells) at the lowerconcentrations (25 mg/l and 50 mg/l), but a good differential pattern ofcolor change at 75 mg/l.

Although the resazurin concentration may need to be adjusted dependingupon the organism tested, the use of resazurin as a color indicator mayprovide additional valuable information to characterize organisms at thespecies or strain level.

In the course of these experiments, it was also observed that pigmentsproduced by some actinomycetes in the various carbon sources tended tocreate very distinct and unique patterns. The unexpected observation wasmade that pigment production was enhanced by using a gel-formingsubstance in the inoculant.

Thus, different color patterns were obtained with the differingresazurin dye concentrations in combination with the natural pigmentsproduced. For example, at 50 mg/l resazurin, M. chalcea produced a rangeof color intensities from colorless to light pink to bright pink andpurple. S. hygroscopicus produced a range of colors from yellow andorange, to colorless, pink and blue. Other species exhibited otherdistinct color patterns in the wells. This additional informationrelated to pigmentation and resazurin dye reduction, may be valuable totaxonomists and others interested in characterizing specific strainsand/or species of actinomycetes.

EXAMPLE 7 Use of Alternative Gelling Agents

Other gelling agents were tested in this Example. In addition toGelrite™, alginic acid, carrageenan type I, carrageenan type II, andpectin were tested for their suitability in the present invention. Allof these compounds are commercially available from Sigma.

Of these compounds, pectin was found to be unsuitable when tested byadding 1% pectin to SF-P MicroPlates™. Pectin has a yellowish cast toit, and is therefore not a colorless or clear compound. Furthermore,gelling was dependent upon the presence of sugars in the microplatewells. Because many of the substrates tested in this multitest format donot contain sugars, gelling did not occur uniformly in all wells.

All of these gelling agents with the exception of pectin, were testedwith the eleven actinomycetes listed in Table 2. The same MicroPlates™(SF-P), incubation time and temperature, as described in Example 5above, were used. The only variables were the different gelling agentsand varying concentrations of these agents.

The optimal viscosity and performance for each gelling agent wasdetermined. Optimal viscosity and performance was achieved at 1% alginicacid; 0.2% was optimum for both types of carrageenan; and 0.4% wasoptimum for Gelrite™. All of these gelling agents were also diluted tohalf the above concentrations and found to be useful even at these lowerconcentrations.

Overall, the results for Gelrite™ and carrageenan types I and II weresimilar, and the difference in gel concentration did not affect theresults significantly. However, the results for alginic acid were not asclearcut when the MicroPlates™ were observed by eye, as compared to theuse of an automatic plate reader (e.g., Biolog MicroStation Reader™,Biolog). Indeed, when read by eye, the results with alginic acid weresomewhat inferior to those obtained with Gelrite™. Carrageenan type IIwas slightly better than type I and it was also comparable to or betterthan Gelrite™. Surprisingly, the carrageenan type II functions aseffectively as the Gelrite™, although the carrageenan does not form arigid gel. This indicates that it is not necessary that a rigid gel beformed in order for the beneficial effects of these colloidal gellingagents to be observed.

EXAMPLE 8 Testing of Other Bacterial Species

In addition to the actinomycetes, the present invention is also suitablefor the rapid characterization of numerous and diverse organisms, suchas those listed in Table 3. The gram-negative bacteria tested covered arange of genera and tribes, including Pseudomonas cepacia, Providenciastuartii, Neisseria lactamica, Xanthomonas maltophilia, Vibriometschnikovii, Cedecea neteri, and Escherichia coli. Variousgram-positive bacteria were also tested, including Rhodococcus equi andStaphylococcus aureus.

These organisms were tested basically as described in Example 5 above,with GN MicroPlates™ (Biolog) used to test the gram-negative organisms,and GP MicroPlates™ (Biolog) used to test the gram-positive organisms.In addition, ES MicroPlates™ (Biolog) were also tested with some of thegram-negative species. Inoculation in 0.4% Gelrite™ was compared toinoculation in 0.85% saline. The inoculation densities used were thosenormally recommended for these MicroPlate™ test kits (55% transmittancefor the gram-negative organisms, and 40% for the gram-positiveorganisms). Following inoculation of the MicroPlates™ with 150 μlsuspensions of organisms in either saline or Gelrite™ per well, theMicroPlates™ were incubated at 35° C. for 16-24 hours.

All of these organisms performed well in the gel, with most producingbetter results in gel than in saline. For example, in the ESMicroPlate™, E. coli produced 43 positive reactions within 24 hours whenthe gel was used, but only 36 positive reactions when saline was used. Acorrect identification of C. neteri was obtained after only 4 hours ofincubation in the Gelrite™, whereas overnight incubation was requiredfor saline. Thus, a correct identification of this organism is possiblein a much shorter time period than the 24 hour incubation usuallyrequired for traditional testing methods.

In contrast to conventional biochemical testing materials and methodstraditionally used, the present invention often achieves a definitiveidentification in a significantly shorter time period.

EXAMPLE 9 Testing of Eukaryotic Microorganisms--Yeasts

This experiment was designed to determine the suitability of the presentinvention for use in identification of eukaryotic microorganisms, suchas yeasts. In this experiment, two types of reactions were observed toestablish a metabolic pattern: a) assimilation reaction tests which arebased on turbidity increases due to carbon utilization by the organisms;and b) oxidation tests, which also test for carbon utilization, butwhich detect utilization via a redox color change of the organismsuspension.

In this experiment, yeasts were first grown on BUY Agar (Biolog) a solidagar medium, and harvested from the agar surface as described in Example2 above. The organisms included in this example are listed in Table 3(D. ovetensis, C. laurentii, C. terreus, K. marxianus, S. cerevisiae,and W. saturnus). Biolog YT MicroPlates™ (available commercially fromBiolog) were then inoculated with an inoculum having an opticaltransmittance of 50%, in either water or 0.4% Gelrite™. Each well of theYT MicroPlate™ was inoculated with 100 μl of either the water or 0.4%Gelrite™ suspension of organisms. Thus, there were two sets of 6MicroPlates™ each. The inoculated MicroPlates™ were incubated at 27° C.,and the results observed at 24, 48, and 72 hours of incubation.

With the oxidation tests, in most cases, the color changes developedmore rapidly in the plates with Gelrite™ used as the inoculant, comparedto the plates with water as the inoculant. For example, D. ovetensis, W.saturnus, K. marxianus, and C. laurentii gave stronger reactions at 48hours with Gelrite™. In contrast, S. cerevisiae and C. terreus gavestronger reactions at 48 hours with water.

With the assimilation tests, in all cases the Gelrite™ was superior orequivalent to the water inoculant. The data shown in the Tables belowclearly demonstrate that more positive (+) and borderline (b) reactionswere obtained overall, when Gelrite™ was used.

                  TABLE 5                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                     After One Day of Incubation                                                                   Water   Gelrite ™                                          Organsim        (+/b)   (+/b)                                                 ______________________________________                                        D. ovetensis     0/5    17/7                                                  K. marxianus    14/3    16/9                                                  W. salurnus      9/7    40/9                                                  C. ferreus A     4/14   33/3                                                  C. laurentii    61/5    67/8                                                  S. cerevisiae A 24/5    22/2                                                  ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                     After Two Days of Incubation                                                                  Water   Gelrite ™                                          Organsim        (+/b)   (+/b)                                                 ______________________________________                                        D. ovetensis     9/2    22/2                                                  K. marxianus    14/5    39/4                                                  W. saturnus     23/7    46/5                                                  C. terreus A    21/7    45/4                                                  C. laurentii    65/0    77/3                                                  S. cerevisiae A 24/6    24/0                                                  ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Positive (+) and Borderline (b) Reactions                                     After Three Days of Incubation                                                                Water   Gelrite ™                                          Organsim        (+/b)   (+/b)                                                 ______________________________________                                        D. ovetensis    21/9    23/7                                                  K. marxianus    27/5    43/7                                                  W. saturnus     48/6    52/3                                                  C. terreus A    20/8    58/5                                                  C. laurentii    68/6    78/5                                                  S. cerevisiae A 24/8    24/2                                                  ______________________________________                                    

In these experiments, the surprising observation was made that someorganisms could be identified faster due to better growth (i.e., growththat appeared much more rapidly and at a greater density), in the platewith the Gelrite™, as compared to the plate with water. For example,Dipodascus ovetensis developed a metabolic reaction pattern sufficientfor correct identification after 24 hours of incubation in the Gelrite™plate, while 48 hours of incubation was required to make the properidentification in the water plate.

In addition, many of the limitations and deficiencies of currentlycommercially available yeast identification systems, such as the Minitek(BBL), API 20C (API), expanded Uni-Yeast-Tek System (Flow), and Vitek(Biomerieux) were overcome or avoided in the present example (see e.g.,G. A. Land (ed.), "Mycology," in H. D. Isenberg (ed.), ClinicalMicrobiology Procedures Handbook, American Society for Microbiology, inparticular "Commercial Yeast Identification Systems," pp. 6.10.1 through6.10.5, [1994]). For example, in the Vitek system, heavily encapsulatedyeasts and isolates with extensive mycelial growth are sometimesdifficult to suspend. As indicated above, this limitation is avoided bythe present invention, allowing for reliable and reproducible testingprocedures and systems. In summary, the Gelrite™ was shown to be clearlysuperior to water for the rapid identification of eukaryoticmicroorganisms.

EXAMPLE 11 Testing of Eukaryotic Microorganisms--Molds

This experiment was designed to determine the suitability of the presentinvention for use in identification of eukaryotic microorganisms, suchas molds.

In this experiment, the molds were first grown on modifiedSabouraud-Dextrose agar (commercially available from various sources,including Difco). This medium is prepared by thoroughly mixing dextrose(20 g/l), agar (20 g/l), and neopeptone (1 g/l) in 1 liter ofdistilled/deionized water. Heat is applied, until the mixture boils. Themedium is autoclaved for 15 minutes at 15 psi (121° C.). After cooling,the medium is distributed into petri plates.

The organisms included in this example are listed in Table 3 (P.notatum, P. chrysogenum, R. pusillus, A. niger and T. mentagrophytes).After they were grown on Sabouraud-Glucose agar, an inoculum wasprepared as described in Example 1. YT and SP-F MicroPlates™ (Biolog)were then inoculated with a 1:10 dilution of a starting inoculum havingan optical transmittance of 70%, in water, 0.2% carrageenan type II, or0.4% Gelrite™.

Each well of the SF-P MicroPlates™ was inoculated with 100 μl oforganisms suspended in either water, 0.2% carrageenan type II, or 0.4%Gelrite™. For the YT plates, 100 μl of organisms suspended in eitherwater, or 0.4% Gelrite™ were used to inoculate the wells. The inoculatedMicroPlates™ were incubated at 25° C., and the results observed by eyeand using a MicroStation Reader™ (Biolog) at 24 hour increments for atotal of 4 days of incubation.

In nearly all cases, the turbidity changes developed more rapidly in theplates with carrageenan or Gelrite™ used as the inoculant, compared tothe plates with water as the inoculant. The data shown in the Tablesbelow clearly demonstrate that for most organisms, more positive (+) andborderline (b) reactions were obtained overall, when carrageenan orGelrite™ was used, as compared to water. The results in these Tables arethose observed with the MicroStation Reader™ (Biolog).

It was also observed that the improvement in the results using Gelrite™or carrageenan as the gelling agent were sometimes more apparent whenthe test results were read visually, rather than by a machine (Biolog'sMicroStation Reader™). This was the case with T mentagrophytes, wherethe improved results obtained with carrageenan were in fact, alsoobtained with Gelrite™, although the reader did not detect thisaccurately at 72 hours. However, with longer incubation periods (e.g.,4-5 days), the visual and machine readings agree very well in nearly allcases.

                  TABLE 8                                                         ______________________________________                                        Positive(+)/Borderline (b) Reactions                                          After 72 Hours of Incubation in SF-P MicroPlates ™                                      Carrageenan Gelrite ™                                                                           Water                                       Organism     (+/b)       (+/b)    (+/b)                                       ______________________________________                                        P. notatum   54/11       52/14    47/11                                       P. chrysogenum                                                                             56/13       54/11    50/17                                       R. pusillus   4/13       5/5      2/6                                         A. niger     23/17       29/12    17/10                                       T. mentagrophytes                                                                          16/12       3/6      5/1                                         ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Positive(+)/Borderline (b) Reactions                                          After 72 Hours of Incubation in YT MicroPlates ™                                             Gelrite ™                                                                           Water                                              Organism          (+/b)    (+/b)                                              ______________________________________                                        P. notatum        78/5     67/4                                               P. chrysogenum    81/1     75/10                                              R. pusillus        17/22   13/26                                              A. niger          78/2     51/11                                              T. mentagrophytes  2/1     2/1                                                ______________________________________                                    

EXAMPLE 12 Antimicrobial Susceptibility Testing

In this Example, the suitability of a gel matrix for use inantimicrobial susceptibility testing was investigated. Two organisms,Staphylococcus aureus (ATCC #29213) and Escherichia coli (ATCC#25922)were tested against a panel of three antimicrobial agents: ampicillin,kanamycin, and tetracycline. All three antimicrobials were obtained fromSigma. Biolog's MT MicroPlates™ (Biolog), were used with 12.5 μl of a10% glucose solution added to each well. Kanamycin and tetracycline weredissolved in sterile water. Ampicillin was dissolved in phosphate buffer(pH 8.0)(0.1 M/l NaH₂ PO₄.H₂ O). For each antimicrobial agent, adilution series ranging from 0.25 μg/ml to 32 μg/ml final concentration,was prepared. A 15 μl aliquot of each dilution was pipetted into thewells of the MicroPlates™, with water used to dilute the kanamycin andtetracycline, and phosphate buffer (pH 6.0)(0.1 M/l NaH₂ PO₄.H₂ O) usedto dilute the ampicillin. For each MicroPlate™, a row of eight wellswithout antimicrobials was used as a control. In the MT MicroPlates™,tetrazolium is included as a color indicator. Unlike the actinomycetes,the most commonly isolated gram-negative and gram-positive bacteria arenot significantly inhibited by the presence of tetrazolium in theseMicroPlates™.

In addition to the MT MicroPlates™, Biolog's SF-N MicroPlates™ (GNMicroPlates™ without tetrazolium), and SF-P MicroPlates™ (GPMicroplates™ without tetrazolium) were tested (all of these plates wereobtained from Biolog). E. coli was inoculated into the SF-NMicroPlates™, and S. aureus was inoculated into the SF-P MicroPlates™.In these MicroPlates™, 25 mg/l of resazurin was added as a colorindicator as an alternative to tetrazolium. In addition, 12.5 μl of 10%glucose solution and 15 μl of each antimicrobial dilution were added toeach well, as described in the paragraph above.

All of the wells in all of the MicroPlates™ were inoculated with 100 μlof a very light suspension (e.g., a 1:100 dilution of a 55%transmittance suspension of E. coli, and a 1:100 dilution of a 40%transmittance suspension of S. aureus), and incubated overnight at 35°C.

For each organism and each MicroPlate™, 0.85% saline and 0.4% Gelrite™were compared, by looking visually for the lowest antimicrobialconcentration that inhibited dye (tetrazolium or resazurin) reduction.The minimum inhibitory concentration (MIC) for each organism wasdetermined after 18 hours of incubation at 35° C. The MIC values foreach organism, as determined from these experiments, are provided in theTables below.

                  TABLE 10                                                        ______________________________________                                        MIC Determinations for E. coli                                                in MT MicroPlates ™ Containing Tetrazolium                                 and SF-N MicroPlates ™ Containing Resazurin                                          Anitmicrobial                                                       Diluent     Ampicillin Kanamycin Tetracycline                                 ______________________________________                                        Saline      1-2        16-32     0.5-1                                        Gelrite ™                                                                              2-4         8-16     0.5-1                                        NCCLS       2-8        1-4         1-4                                        Expected Result                                                               ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        MIC Determinations for S. aureus                                              in SF-P MicroPlates ™ Containing Resazurin                                           Anitmicrobial                                                       Diluent     Ampicillin Kanamycin Tetracycline                                 ______________________________________                                        Saline      1-4        16-32     0.25-2                                       Gelrite ™                                                                              1-2        16-32     0.25-1                                       NCCLS       0.25-1     1-4       0.25-1                                       Expected Results                                                              ______________________________________                                    

As shown in these tables, the results in the Gelrite™ agreed with theresults obtained with saline as an inoculant within one two-folddilution. This is considered satisfactory according to the NationalCommittee on Clinical Laboratory Standards (NCCLS) guidelines (see e.g.,J. Hindler (ed.), "Antimicrobial Susceptibility Testing," in H. D.Isenberg (ed.), Clinical Microbiology Procedures Handbook, AmericanSociety for Microbiology, pp. 5.0.1 through 5.25.1, [1994]). In oneinstance, the MIC was slightly lower in saline as compared to Gelrite™.In three instances, the MIC's were slightly lower in Gelrite™, than insaline. Thus, the present invention provides a novel and usefulalternative method for determination of antimicrobial sensitivities ofmicroorganisms. Another advantage of this invention is that the test maybe conducted in a format that cannot be accidentally spilled.

EXAMPLE 13 Synthesis of Redox Purple

In this Example, the redox indicator referred to as "Redox Purple" wassynthesized for use in the present invention. In this Example, themethod of Graan et al. (T. Graan, et al., "Methyl Purple, anExceptionally Senstive Monitor of Chloroplast Photosystme I Turnover:Physical Properties and Synthesis," Anal Biochem., 144:193-198 [1985])was used with modifications. This synthesis is shown schematically inFIG. 5 and the Roman numerals (i.e. I,II,III,IV and V) used in thisExample refer to those shown in FIG. 5. Unless otherwise indicated, thechemicals used in this Example were obtained from commercial sourcessuch as Sigma.

Briefly, the benzoquinone-4-chloroimide (FIG. 5, II) was produced bydissolving 5 g 4-aminophenol (FIG. 5, I) in 1 N aqueous HCl (75 mL) (0°C.), followed by the addition of 200 mL sodium hypochlorite (NaClO, 5%w/v) to produce a chloroimide derivative shown in FIG. 5, Panel A. Inthis reaction, the solution was continuously stirred and the temperaturemaintained below 4° C. during addition of the sodium hypochlorite. Afterstirring at room temperature for 12 hours, the yellow to orange coloredproduct was isolated by filtration, washed with cold distilled water anddried in air and in vacuo. In this step, the product was vacuum filteredusing a Buchner funnel, washed with a minimal amout of ice-cold water(approximately 30 ml) in the funnel, dried in air for approximately 24hours, and dried overnight in a vacuum dessicator.

The synthesis of 1-(3-hydroxyphenyl)-ethanol (FIG. 5, IV) was performedimmediately prior to its use, by the reduction of 5 g1-(3-hydroxyphenyl)-ethanone (available as m-hydroxyacetophenone fromTokyo Kasei kogyo Co., Ltd. Fukaya, Japan, with TCI America, inPortland, Oreg., being the U.S. distributor) (FIG. 5, III) in water (300mL) with sodium borohydride (NaBH₄, 1.5 g), as shown in FIG. 5, Panel B.The reaction was warmed as necessary to dissolve the starting materialand stirred until the evolution of H₂ ceased (approximately 1 hour). ThepH was decreased to 2.0 (i.e., with concentrated HCl) to remove excessborohydride, followed by addition of 150 ml saturated sodium borate.

The synthesis of redox purple was initiated by addition of thechloroimide derivative (II) to the freshly prepared solution of1-(3-hydroxyphenyl)-ethanol (IV), in borate buffer (Na₂ B₄ O₇ /H₃ BO₃).Sodium arsenite (NaAsO₂, 10 g) (Sigma) was added to the reactionsolution, in order to promote the formation of the indophenol, as wellas minimize the occurrence of side reactions. This reaction solution wasstirred at room temperature for 2 hours, during which the blue color ofthe indophenol (FIG. 5, V) appeared. The reaction mixture was thenallowed to sit at room temperature for 7-8 days, during which theclosure of the heterocyclic ring was allowed to occur due to formationof an oxymethylene group bridge between the two phenolic residues of thequinone-imide. The ring closure was accompanied by a change in thesolution color to a dark purple.

The reaction mixture was filtered and the precipitate washed withminimal cold water as described above. The filtrate was saturated withan excess of solid sodium chloride (approximately 100 g), the solutionwas decanted off the excess salt on the bottom of the container, and thesolution extracted with diethylether (5×100 mL) until no moreorange-colored material was removed from the aqueous phase. Vigorousshaking of the ether and aqueous phases was avoided, as this was foundin some experiments to result in formation of an intractable emulsion.The combined ether layers were back-extracted with 70 mM aqueous sodiumcarbonate solution (25 mL), the pH of the sodium carbonate solutionreduced to 4.5 with glacial acetic acid, and the resulting mixturerefrigerated overnight at 4° C. The redox purple precipitated as thefree acid. Additional redox purple was obtained by acidifying theoriginal aqueous phases with glacial acetic acid (pH 4.5) and repeatingthe above purification. The total yield obtained by this synthesismethod was approximately 25%.

The purity of the redox purple synthesized according to this method was95-98%, as determined by thin-layer chromatography, a method that iswell know in the art (A. Braithwaite and F. J. Smith, in"Chromatographic Methods" Chapman and Hall [eds.], London [1985], pp.24-50.). It was found that the redox purple compound was not verysoluble in water as the free acid, but was quite soluble in slightlybasic solutions (e.g., 1 N NaHCO₃), or in organic solvents (e.g.methanol, ethanol, dimethyl sulfoxide [DMSO], dimethyl formamide [DMF],etc.). The compound was observed to be a deep purple color (i.e., ofapproximately 590 nm as an absorption wavelength) in basic solution andan orange-red color (470 nm) in acidic solution. It is contemplated thatanalogous derivatives of the reagent (e.g., alkali salts, alkylO-esters), with modified properties (e.g., solubility, cellpermeability, toxicity, and/or modified color(s)/absorption wavelengths)will be produced using slight modifications of the methods describedhere. It is also contemplated that various forms of redox purple (e.g.,salts, etc.), may be effectively used in combination as a redoxindicator in the present invention.

EXAMPLE 14 Redox Purple and E. coli Identification

In this Example, redox purple was used as the redox indicator in thetest system E. coli 287 (ATCC #11775) was cultured overnight at 35° C.,on TSA medium supplemented with 5% sheep blood. A sterile, moistened,cotton swab was used to harvest colonies from the agar plate and preparesix identical suspensions of organisms in glass tubes containing 18 mlof 0.85% NaCl, or 0.2% carrageenan type II. The cell density wasdetermined to be 53-59% transmittance. One saline and one carrageenansuspension were used to inoculate Biolog GN Microplates™, with 150 μ laliquots placed into each well. The wells of this plate containtetrazolium violet as the redox indicator. Two ml of a 2 mM solution ofredox purple (sodium salt)(prepared as described in Example 13), or twoml of a 2 mM solution of resazurin (sodium salt) were added to the othertubes, to produce a final dye concentration of 200 μM. These suspensionswere used to inoculate Biolog SF-N Microplates™. As with the GNMicroplates™, aliquots of 150 μl were added to each well in the plates.The SF-N Microplates™ are identical to the GN MicroPlates™, with theexception being the omission of tetrazolium violet from the wells of theSF-N plates. The inoculated plates were incubated at 35° C. forapproximately 16 hours. The plates were then observed and the colors ofthe well contents recorded.

For the 0.85% NaCl and 0.2% carrageenan suspensions inoculated into theSF-N Microplate™, positive results were obtained for all three redoxindicators (i.e., redox purple, tetrazolium violet, and resazurin) inwells containing the following carbon sources: dextrin, tween-40,tween-80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose,D-fructose, L-fucose, D-galactose, α-D-glucose, α-D-lactose, maltose,D-mannitol, D-mannose, D-melibiose, β-methyl-D-glucoside, L-rhamnose,D-sorbitol, D-trehalose, methyl pyruvate, mono-methyl succinate, aceticacid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid,D-glucuronic acid, α-ketobutyric acid, D,L-lactic acid, propionic acid,succinic acid, bromosuccinic acid, alaninamide, D-alanine, L-alanine,L-alanyl-glycine, L-asparagine, L-aspartic acid, glycyl-L-aspartic acid,glycyl-L-glutamic acid, D-serine, L-serine, inosine, uridine, thymidine,glycerol, D,L-α-glycerol phosphate, glucose-1-phosphate, andglucose-6-phosphate.

For the 0.85% NaCl and 0.2% carrageenan suspensions, negative resultswere obtained for all three redox indicators (i.e. redox purple,tetrazolium violet, and resazurin) in wells containing the followingcarbon sources: α-cyclodextrin, adonitol, D-arabitol, cellobiose,i-erythritol, xylitol, citric acid, D-glucosaminic acid,β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxyphenylaceticacid, itaconic acid, α-ketovaleric acid, malonic acid, quinic acid,sebacic acid, L-histidine, hydroxy L-proline, L-leucine, andD,L-carnitine. The negative control wells containing water, instead of acarbon source were also negative for all three redox indicators.

For glycogen, D-psicose, succinamic acid, and glucuronamide, negativeresults were obtained with both the 0.85% NaCl and carrageenansuspensions with redox purple. However, positive results were obtainedfor both suspensions with tetrazolium violet and resazurin.

For gentiobiose, m-inositol, cis-aconitic acid, L-phenylalanine,L-pyroglutamic acid, phenylethylamine, putrescine, 2-amino ethanol, and2,3-butanediol negative results were obtained with both the 0.85% NaCland carrageenan suspensions with redox purple and tetrazolium violet.However, positive/negative results were obtained with the 0.2%carrageenan suspension in resazurin, while the resazurin result with the0.85% NaCl was negative.

For lactulose, D-raffinose, formic acid, α-hydroxybutyric acid,L-glutamic acid, and L-proline, negative results were observed with the0.85% NaCl suspension tested with redox purple, although the remainingresults were positive.

For sucrose and L-ornithine, negative results were obtained for both the0.85% NaCl and 0.2% carrageenan suspensions tested with redox purple andtetrazolium violet. However, a negative result was observed for the0.85% NaCl suspension tested with resazurin and a positive result wasobserved for the 0.2% carrageenan suspension.

For turanose, both the 0.85% NaCl and 0.2% carrageenan suspensions werenegative when tested with redox purple, while the results for bothtested with tetrazolium violet were equivocal (+/-), the result for the0.85% NaCl suspension tested with resazurin was also equivocal (+/-),and the result for the 0.2% carrageenan tested with resazurin waspositive.

For α-ketoglutaric acid, negative results were observed for both the0.85% NaCl and 0.2% carrageenan suspensions tested with redox purple andtetrazolium violet, while positive results were observed for bothsuspensions tested with resazurin.

For D-saccharic acid, negative results were observed for both the 0.85%and 0.2% carrageenan suspensions tested with redox purple, while theresult with tetrazolium violet was equivocal (+/-) for 0.85% NaCl andnegative for carrageenan, and the result with resazurin was negative forthe 0.85% NaCl and positive for 0.2% carrageenan suspensions.

For L-threonine, equivocal (+/-)results were observed for 0.2%carrageenan suspensions tested with redox purple and tetrazolium violet,while the result with resazurin was positive. For the 0.85% NaClsuspension, the result was negative for redox purple, and positive fortetrazolium violet and resazurin.

For γ-aminobutyric acid and urocanic acid, negative results wereobserved for both the 0.85% NaCl and 0.2% carrageenan suspensions testedwith redox purple and tetrazolium violet, while equivocal (+/-)resultswere observed with 0.85% NaCl, and positive results were observed withthe 0.2% carrageenan.

In the inoculated GN Microplate™ (containing tetrazolium violet), thewells corresponding to the carbon sources utilized by E. coil 287 becameeither a light or dark purple, while the wells corresponding to thecarbon sources not utilized by this organism remained colorless. Incontrast, in the inoculated SF-N Microplate™ (containing redox purple),the color pattern was virtually reversed. For negative wells with redoxpurple, a blue to purple (i.e.,blue-purple, purple-tinged blue, orviolet) color was observed. In the SF-N Microplate™ plate, the wellscorresponding to carbon sources utilized by this organism were lightblue or were colorless, while the wells containing carbon sources notutilized by this organism remained dark blue. The color patterns wereeasily read and analyzed. Thus, the redox purple was shown to work in amanner that appears to be equivalent to tetrazolium violet for detectingcarbon source utilization by bacteria. However, there were three colorsobserved with the plates which included resazurin (i.e., blue, pink andcolorless), making the redox purple a more useful redox indicator, asthere was less ambiguity in the reading of the results.

The observation that none of the wells with redox purple was orange wasvery surprising, as the literature describing this compound indicatedthat there was an intermediate stage in the reduction of the dye whichwas expected to be reduced through the color progression of blue toorange to colorless. This two-stage reduction is in contrast to thetypical reaction observed with resazurin, which gives blue, pink, andcolorless wells when analyzed in a like manner. The side-by-side datafor the resazurin in this experiment, as well as other tests, confirmsthat it does form three colors. The degree to which the results of thevarious plates were in agreement are shown in the following Table.

                  TABLE 12                                                        ______________________________________                                        Comparison of Redox Purple and Resazurin                                      with Tetrazolium Violet                                                                          Number                                                                        of Wells With Same                                         Solution                                                                            Dyes Compared                                                                              Result (96 Wells/Plate)                                                                      % Agreement                                 ______________________________________                                        Saline                                                                              Redox Purple/                                                                              85/96          88.5                                              Tetrazolium Violet                                                      Gel   Redox Purple/                                                                              92/96          95.8                                              Tetrazolium Violet                                                      Saline                                                                              Resazurin/   95/96          99.0                                              Tetrazolium Violet                                                      Gel   Resazurin/   94/96          94.8                                              Tetrazolium Violet                                                      ______________________________________                                    

The oxidized form of redox purple spectrally matches the reduced form oftetrazolium violet (i.e., with a maximum absorbance at 590 nm). This mayprovide an advantage, as detection methods such as spectrophotometrysettings may be used interchangeably with tetrazolium violet and redoxpurple.

EXAMPLE 15 Redox Purple and Identification of Fungi

In this Example, Aspergillus niger, Penicillium chrysogenum, andTrichoderma harzianum were tested using the redox purple redoxindicator.

First, the above named organisms were tested using the GN Microplate.However, none of these organisms reduced the tetrazolium violet in thewells of the plate. Thus, redox purple was investigated for use as analternative dye.

T. harzianum DAOM 190830 was cultured for seven days at 26° C. on maltextract agar (Difco). A sterile, moistened cotton swab was used toharvest conidia from the culture and prepare a suspension in 16 ml of0.25% Gelrite™. The cell density was determined to be 75% transmittance.A 2 ml aliquot of a 2 mM solution of redox purple was added to thesuspension, along with 2 ml of 1 M triethanolamine-SO₄, pH 7.3. Thefinal concentration of redox purple was 200 μM, and the finalconcentration of triethanolamine-SO₄ was 100 mM. The final suspensionwas mixed well and used to inoculate the wells of a Biolog SF-NMicroplate™. In this Example, 100 μl of the suspension was added to eachwell. The inoculated SF-N Microplate™ was incubated at 30° C. forapproximately 24 hours, and observed.

For each carbon source utilized by the organism, the content of thewells was colorless. For each carbon source not utilized by theorganism, the content of the wells was blue. In this Example, for thisculture, positive results were obtained in the wells containing dextrin,glycogen, tween-40, tween-80, N-acetyl-D-glucosamine, L-arabinose,D-arabitol, cellobiose, i-erythritol, D-fructose, L-fucose, D-galactose,gentiobiose, α-D-glucose, D-mannitol, D-mannose, D-melibiose,β-methyl-D-glucoside, D-sorbitol, D-trehalose, methyl pyruvate,mono-methyl succinate, citric acid, D-galacturonic acid,β-hydroxybutyric acid, α-ketoglutaric acid, quinic acid, sebacic acid,succinic acid, bromo succinic acid, succinamic acid, L-alanine,L-alanyl-glycine, L-asparagine, L-glutamic acid, gylcyl-L-glutamic acid,L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine,γ-amino butyric acid, inosine, and glycerol.

From the above Examples, it is clear that the present inventionrepresents an unexpected and much improved system for the rapidbiochemical testing of microorganisms, in many uses and formats (orconfigurations) and in particular, provides a major advance in thetesting of actinomycetales and other spore-forming microorganisms. Theresults are highly surprising in view of the obligate aerobic nature ofmost of these organisms. Using the novel approach of embedding theorganisms in a gel matrix, the biochemical test reactions are disperseduniformly throughout the testing well, providing an easy to readindicator of organism growth and metabolism. In addition, both automatedand manual systems may be used in conjunction with the presentinvention. For example, the results may be observed visually (i.e., byeye) by the person conducting the test, without assistance from amachine. Alternatively, the results may be obtained with the use ofequipment (e.g., a microplate reader) that measures transmittance,absorbance, or reflectance through, in, or from each well of a multitestdevice such as microplate or microcard.

In summary, the present multitest gel-matrix invention provides numerousadvances and advantages over the prior art, including: (1) much greatersafety, as there is no spillage, nor aerosolization of cells, mycelia,nor spores, while performing or inoculating test wells; (2) fasterbiochemical reactions are produced, giving final results hours or daysearlier than commonly used methods; (3) more positive biochemicalreactions are obtained, giving a truer picture of the microorganisms'metabolic characteristics; (4) darker, more clear-cut biochemicalreactions and color changes are obtained; (5) more uniform color and/orturbidity are obtained, as the cells, mycelia, and/or spores do notsettle and clump together at the bottom of the wells, nor do they adhereto the sides of the wells; (6) the reactions are much easier to observevisually or with optical instruments (e.g., the Biolog MicroStationReader™); and (7) the overall process for performing multiple tests isextremely simple and efficient, requiring very little labor on the partof the microbiologist. All of these advantages enhance the speed andaccuracy of scoring test results in studies to characterize and/oridentify microorganisms.

What is claimed is:
 1. A kit for characterizing microorganisms, wherein said kit comprises redox purple, and one or more test substrates.
 2. The kit of claim 1, wherein said test substrates are selected from the group consisting of carbon sources and antimicrobials.
 3. The kit of claim 1 further comprising one or more gel-initiating agents.
 4. The kit of claim 3, wherein said gel initiating agent comprises cationic salts.
 5. The kit of claim 1, further comprising one or more gelling agents.
 6. The kit of claim 5, wherein said gelling agent is selected from the group consisting of agar, Gelrite™, carrageenan, and alginic acid.
 7. The kit of claim 1, further comprising a suspension of microorganisms.
 8. The kit of claim 1, further comprising a testing means.
 9. The kit of claim 1, wherein said testing means comprises a microplate.
 10. A kit for characterizing microorganisms wherein said kit comprises redox purple and one or more gelling agents.
 11. The kit of claim 10, wherein said gelling agent is selected from the group consisting of Gelrite™, carrageenan, and alginic acid.
 12. The kit of claim 10, further comprises one or more gel-initiating agents.
 13. The kit of claim 12, wherein said gel-initiating agent comprises cationic salts.
 14. The kit of claim 12, further comprising one or more test substrates.
 15. The kit of claim 14, wherein said test substrates are selected from the group consisting of carbon sources and antimicrobials.
 16. The kit of claim 10, further comprising a suspension of microorganisms.
 17. The kit of claim 10, further comprising a testing means.
 18. The kit of claim 17, wherein said testing means comprises a microplate.
 19. A kit for identification of microorganisms, wherein said kit comprises redox purple and one or more test substrates.
 20. The kit of claim 19, wherein said test substrates are selected from the group consisting of carbon sources and antimicrobials.
 21. The kit of claim 19, further comprising one or more gel initiating agent.
 22. The kit of claim 21, wherein said gel-initiating agent comprises cationic salts.
 23. The kit of claim 19, further comprising one or more gelling agents.
 24. The kit of claim 19, wherein said gelling agent is selected from the group consisting of agar, Gelrite™, carrageenan, and alginic acid.
 25. The kit of claim 19, further comprising a suspension of microorganisms.
 26. The kit of claim 19, further comprising a testing means.
 27. The kit of claim 19, wherein said testing means comprises a microplate.
 28. A kit for identification of microorganisms, wherein said kit comprises redox purple and one or more gelling agents.
 29. The kit of claim 28, wherein said gelling agent is selected from the group consisting of Gelrite™, carrageenan, and alginic acid.
 30. The kit of claim 28, further comprises one or more gel-initiating agents.
 31. The kit of claim 30, wherein said gel-initiating agent comprises cationic salts.
 32. The kit of claim 28, further comprising one or more test substrates.
 33. The kit of claim 32, wherein said test substrates are selected from the group consisting of carbon sources and antimicrobials.
 34. The kit of claim 28, further comprising a suspension of microorganisms.
 35. The kit of claim 28, further comprising a testing means.
 36. The kit of claim 35, wherein said testing means comprises a microplate. 